| Assay Method Information | |
| | Determination of Binding Affinity to EBP-7-Dehydrocholesterol Reductase |
| Description: | Compounds were prepared in a 96-well U bottom plate using an Echo550 machine and 10 mM compound DMSO stock solution, followed by an 8-dose 5-fold serial dilutions protocol with final testing compound concentration ranging from 0.06 to 5000 nM, with DMSO back fill to 100 nL/well and n=2. DMSO and Ifenprodil 5 uM wells were added in each plate as 0 and 100% inhibition reference controls with n=8 for each condition. The UniFilter-96 GF/B plates were pre-treated by adding 50 μl/well of 0.3% (v/v) PEI to UniFilter-96 GF/B plates. The plates were sealed and incubated at 4° C. for 3 hrs. Then, the plates were washed with ice-cold assay buffer 3 times. The radioligand binding assay was prepared by adding assay buffer diluted hEBP-DHCR7 membrane at 66.7 μg/ml x 150 μl/well into the 96-well compound plate to reach 10 μg membrane per well. Then, the assay buffer diluted [3H]-Compound 118A* was added at 25 nM×50 μl/well. Following this, the plate was centrifuged at 1000 rpm for 30 secs. The plate was then sealed and agitated at 600 rpm at 22° C. for 5 min, and then incubated at 22° C. for 3 hrs. The incubation was stopped by transferring the binding solution to the pre-treated UniFilter-96 GF/B plate, vacuum filtrated, and then washed four times with ice-cold assay buffer. Following this, the plates were dried at 37° C. for 45 min. The plates were then sealed at the bottom. 40 μl/well of scintillation cocktail was added to the plates. A MicroBeta2 microplate counter was then used to read the plate and analyze the data. For reference and test compounds, the results are expressed as % Inhibition, using the normalization equation: N=100-100×(U−C2)/(C1−C2), where U is the unknown value, C1 is the average of high controls, and C2 is the average value of low controls. The IC50 was determined by fitting percentage of inhibition as a function of compound concentrations with Hill equation using XLfit. |
| Affinity data for this assay | |
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